Loss of SOCS3 in T helper cells resulted in reduced immune responses and hyperproduction of interleukin 10 and transforming growth factor–β1

Suppressor of cytokine signaling (SOCS)3 is a major negative feedback regulator of signal transducer and activator of transcription (STAT)3-activating cytokines. Transgenic mouse studies indicate that high levels of SOCS3 in T cells result in type 2 T helper cell (Th2) skewing and lead to hypersensitivity to allergic diseases. To define the physiological roles of SOCS3 in T cells, we generated T cell–specific SOCS3 conditional knockout mice. We found that the mice lacking SOCS3 in T cells showed reduced immune responses not only to ovalbumin-induced airway hyperresponsiveness but also to Leishmania major infection. In vitro, SOCS3-deficient CD4+ T cells produced more transforming growth factor (TGF)-β1 and interleukin (IL)-10, but less IL-4 than control T cells, suggesting preferential Th3-like differentiation. We found that STAT3 positively regulates TGF-β1 promoter activity depending on the potential STAT3 binding sites. Furthermore, chromatin immunoprecipitation assay revealed that more STAT3 was recruited to the TGF-β1 promoter in SOCS3-deficient T cells than in control T cells. The activated STAT3 enhanced TGF-β1 and IL-10 expression in T cells, whereas the dominant-negative form of STAT3 suppressed these. From these findings, we propose that SOCS3 regulates the production of the immunoregulatory cytokines TGF-β1 and IL-10 through modulating STAT3 activation

A Cdo–Bnip-2–Cdc42 signaling pathway regulates p38α/β MAPK activity and myogenic differentiation

The p38α/β mitogen-activated protein kinase (MAPK) pathway promotes skeletal myogenesis, but the mechanisms by which it is activated during this process are unclear. During myoblast differentiation, the promyogenic cell surface receptor Cdo binds to the p38α/β pathway scaffold protein JLP and, via JLP, p38α/β itself. We report that Cdo also interacts with Bnip-2, a protein that binds the small guanosine triphosphatase (GTPase) Cdc42 and a negative regulator of Cdc42, Cdc42 GTPase-activating protein (GAP). Moreover, Bnip-2 and JLP are brought together through mutual interaction with Cdo. Gain- and loss-of-function experiments with myoblasts indicate that the Cdo–Bnip-2 interaction stimulates Cdc42 activity, which in turn promotes p38α/β activity and cell differentiation. These results reveal a previously unknown linkage between a cell surface receptor and downstream modulation of Cdc42 activity. Furthermore, interaction with multiple scaffold-type proteins is a distinctive mode of cell surface receptor signaling and provides one mechanism for specificity of p38α/β activation during cell differentiation

Activated Macrophage Survival Is Coordinated by TAK1 Binding Proteins

<div><p>Macrophages play diverse roles in tissue homeostasis and immunity, and canonically activated macrophages are critically associated with acute inflammatory responses. It is known that activated macrophages undergo cell death after transient activation in some settings, and the viability of macrophages impacts on inflammatory status. Here we report that TGFβ- activated kinase (TAK1) activators, TAK1-binding protein 1 (TAB1) and TAK1-binding protein 2 (TAB2), are critical molecules in the regulation of activated macrophage survival. While deletion of <i>Tak1</i> induced cell death in bone marrow derived macrophages even without activation, <i>Tab1</i> or <i>Tab2</i> deletion alone did not profoundly affect survival of naïve macrophages. However, in lipopolysaccharide (LPS)-activated macrophages, even single deletion of <i>Tab1</i> or <i>Tab2</i> resulted in macrophage death with both necrotic and apoptotic features. We show that TAB1 and TAB2 were redundantly involved in LPS-induced TAK1 activation in macrophages. These results demonstrate that TAK1 activity is the key to activated macrophage survival. Finally, in an <i>in vivo</i> setting, <i>Tab1</i> deficiency impaired increase of peritoneal macrophages upon LPS challenge, suggesting that TAK1 complex regulation of macrophages may participate in <i>in vivo</i> macrophage homeostasis. Our results demonstrate that TAB1 and TAB2 are required for activated macrophages, making TAB1 and TAB2 effective targets to control inflammation by modulating macrophage survival.</p></div

TAB1 is required for LPS-activated macrophage survival.

<p>(A) Viability of LPS-treated <i>Tab1^iKO</i> macrophages. <i>Tab1^iKO</i> and control BMDMs were cultured for 8 days with 0.3 µM 4-OHT followed by 3 days 1 µg/ml LPS. Viability was measured by Crystal Violet Assay, and data shown are mean percentages of attached macrophages compared to 8 days treated with vehicle +/− SD of 3 independent experiments. (B) Flow cytometry analysis of <i>Tab1^iKO</i> BMDMs. <i>Tab1^iKO</i> or <i>Tab1^F+ Cre</i> BMDMs were cultured in macrophage medium with 0.3 µM 4-OHT or vehicle (ethanol) for 4 days. All cells including attached and floating cells were collected and stained with annexin V-Pacific Blue and Fixable viability dye eFlour 780, then analyzed on flow cytometer. Events were gated to exclude dead cells and debris, then gated on events positive for annexin V and fixable viability dye compared with unstained controls. Shown is representative figure of 3 independent experiments. (C and D) <i>Tab1^iKO</i> and controls including WT and F+Cre BMDMs were cultured 3 days in 0.3 µM 4-OHT-containing macrophage medium, then 4 days with the addition of 1 µg/ml LPS. (C) Viability Dye-positive cells as a percentage of total cells is shown. (D) Graph shows Annexin V-positive but viability dye-negative cells. Graph represents results of four independent experiments +/− SD.</p

TAK1 is required for macrophage survival.

<p>(A) Western blotting analysis of TAK1, TAB1 and TAB2 in control, <i>Tak1^iKO, Tab1^iKO, Tab2^iKO</i> and <i>diKO</i> BMDMs. Bone marrow cells were cultured in macrophage medium and treated with 0.3 µM 4-OHT or vehicle (ethanol) for 4 days. <i>Tak1^iKO</i> and <i>diKO</i> BMDMs were additionally treated with 50 µM Necrostatin-1 (Nec-1). Anti-β-actin Western blotting was used as a loading control. The numbers beside each panel denote the size and the position of molecular weight markers. (B) Viability of WT, <i>Tak1^iKO, Tab1^iKO, Tab2^iKO</i>, and inducible double-deficient <i>(diKO)</i> BMDMs. Cells were cultured for 8 days with 0.3 µM 4-OHT and stained with 0.1% Crystal Violet. Data are mean percentages of attached macrophages compared to ethanol-treated +/− SD for 3 independent experiments. Asterisks indicate p-values: **  =  P<0.005; ***  =  p<0.0005.</p

LPS activates TAK1 through TAB1 and TAB2.

<p>(A) Western blotting analysis of <i>Tak1^iKO</i> and <i>Tab1Tab2^diKO</i> and control BMDMs. Cells were cultured for 4 days with 0.3 µM 4-OHT in the presence of 50 µM Nec-1, followed by 1 µg/ml LPS treatment for the indicated period of time. Anti-βactin was used as a loading control. Asterisks indicate non-specific bands. (B) <i>Tab1^iKO</i> or control BMDMs were cultured with 0.3 µM 4-OHT for 3 days then treated with 1 µg/ml LPS for the indicated period of time. Whole cell extracts were analyzed by Western blotting using the indicated antibodies. Asterisks indicate non-specific bands. (C) <i>Tab2^iKO</i> or control BMDMs were cultured with 0.3 µM 4-OHT for 3 days then treated with 1 µg/ml LPS for the indicated period of time. Whole cell extracts were analyzed by Western blotting using the indicated antibodies. Asterisks indicate non-specific bands.</p

TAB1 or TAB2 deletion causes RIP1-dependent cell death.

<p>(A) <i>Tab1^iKO</i> or control BMDMs were cultured with 0.3 µM 4-OHT with or without 50 µM Nec-1 for 8 days then treated with 1 µM LPS for 3 days, and viability was measured by Crystal Violet Assay. *  =  p<.05. (B) Viability of <i>Tab2^iKO</i> BMDMs treated with RIP1 inhibitor. <i>Tab2^iKO</i> BMDMs were treated with 0.3 µM 4-OHT for 8 days with our without 50 µM Necrostatin-1 (Nec-1), then treated with 1 µM LPS for one day. Viability was measured by Crystal Violet Assay. *  =  p<.05.</p